Conserved and context-dependent roles for pdgfrb signaling during zebrafish vascular mural cell development.

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.

Peri-arterial specification of vascular mural cells from naïve mesenchyme requires Notch signaling.

Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrblow mesenchymal cells. The emergence of peri-arterial pdgfrbhigh MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrbhigh MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrbhigh MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.

In vivo analysis of cardiomyocyte proliferation during trabeculation.

Cardiomyocyte proliferation is crucial for cardiac growth, patterning and regeneration; however, few studies have investigated the behavior of dividing cardiomyocytes in vivo Here, we use time-lapse imaging of beating hearts in combination with the FUCCI system to monitor the behavior of proliferating cardiomyocytes in developing zebrafish. Confirming in vitro observations, sarcomere disassembly, as well as changes in cell shape and volume, precede cardiomyocyte cytokinesis. Notably, cardiomyocytes in zebrafish embryos and young larvae mostly divide parallel to the myocardial wall in both the compact and trabecular layers, and cardiomyocyte proliferation is more frequent in the trabecular layer. While analyzing known regulators of cardiomyocyte proliferation, we observed that the Nrg/ErbB2 and TGFß signaling pathways differentially affect compact and trabecular layer cardiomyocytes, indicating that distinct mechanisms drive proliferation in these two layers. In summary, our data indicate that, in zebrafish, cardiomyocyte proliferation is essential for trabecular growth, but not initiation, and set the stage to further investigate the cellular and molecular mechanisms driving cardiomyocyte proliferation in vivo.

Tctex-1 augments G protein-coupled receptor-mediated Gs signaling by activating adenylyl cyclase.

Proteins interacting with G protein-coupled receptors (GPCRs) can modulate signal transduction of these receptors. However, the regulatory mechanisms of the interacting proteins are diverse and largely unknown. We have previously shown that Tctex-1 (or DYNLT1) can interact with the parathyroid hormone receptor (PTHR). In the present study, we investigated the role of Tctex-1 in the PTHR signaling and found that Tctex-1 augmented the PTHR-mediated Gs/adenylyl cyclase (AC) pathway by activating AC regardless of the binding to PTHR. Furthermore, Tctex-1 directly bound to AC type 6. These data demonstrate a novel mechanism underlying GPCR/Gs signaling regulated by Tctex-1.

Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.

A New Secretory Peptide of Natriuretic Peptide Family, Osteocrin, Suppresses the Progression of Congestive Heart Failure After Myocardial Infarction.

RATIONALE: An increase of severe ischemic heart diseases results in an increase of the patients with congestive heart failure (CHF). Therefore, new therapies are expected in addition to recanalization of coronary arteries. Previous clinical trials using natriuretic peptides (NPs) prove the improvement of CHF by NPs. OBJECTIVE: We aimed at investigating whether OSTN (osteocrin) peptide potentially functioning as an NPR (NP clearance receptor) 3-blocking peptide can be used as a new therapeutic peptide for treating CHF after myocardial infarction (MI) using animal models. METHODS AND RESULTS: We examined the effect of OSTN on circulation using 2 mouse models; the continuous intravenous infusion of OSTN after MI and the OSTN-transgenic (Tg) mice with MI. In vitro studies revealed that OSTN competitively bound to NPR3 with atrial NP. In both OSTN-continuous intravenous infusion model and OSTN-Tg model, acute inflammation within the first week after MI was reduced. Moreover, both models showed the improvement of prognosis at 28 days after MI by OSTN. Consistent with the in vitro study binding of OSTN to NPR3, the OSTN-Tg exhibited an increased plasma atrial NP and C-type NP, which might result in the improvement of CHF after MI as indicated by the reduced weight of hearts and lungs and by the reduced fibrosis. CONCLUSIONS: OSTN might suppress the worsening of CHF after MI by inhibiting clearance of NP family peptides.

Confined Space Enables Spontaneous Liquid Separation by Molecular Size: Selective Absorption of Alkanes into a Polyolefin Cast Film.

The depletion force has been used to explain phase separation phenomena in colloidal systems. Here, we showed that depletion force can explain not only phase separation of large and small colloidal particles but also preferential absorption of larger molecules from a mixture of large and small molecules in a liquid state. When a polyolefin cast film was immersed in a mixture of long and short normal alkanes, the longer molecules were selectively absorbed into the film. This experimental result was explained from the viewpoint of depletion force. The main finding was the use of confined space to emphasize the separation tendency caused by the force. In general, the increase in entropy may serve as a driving force to mix molecules. However, if sufficiently narrow pores are present, large and small molecules are separated naturally by size as the entropy increases. This finding will lead to size exclusion chromatography of low-mass molecules, similar to gel permeation chromatography of macromolecules. In order to demonstrate the effect of depletion force, we selected and experimented with a system based on a polyolefin isotactic poly(4-methyl-1-pentene) (P4MP1) film and a normal alkane mixture and realized high molecular selectivity. The P4MP1 film we used can be prepared simply by evaporating the solvent from the solution and casting the film. On the basis of the Asakura-Oosawa theory, we concluded that spontaneous and high molecular selectivity is attributed to the depletion force provided by the small sub-nanopores with uniform size in the film.

Cardiomyokines from the heart.

The heart is regarded as an endocrine organ as well as a pump for circulation, since atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were discovered in cardiomyocytes to be secreted as hormones. Both ANP and BNP bind to their receptors expressed on remote organs, such as kidneys and blood vessels; therefore, the heart controls the circulation by pumping blood and by secreting endocrine peptides. Cardiomyocytes secrete other peptides besides natriuretic peptides. Although most of such cardiomyocyte-derived peptides act on the heart in autocrine/paracrine fashions, several peptides target remote organs. In this review, to overview current knowledge of endocrine properties of the heart, we focus on cardiomyocyte-derived peptides (cardiomyokines) that act on the remote organs as well as the heart. Cardiomyokines act on remote organs to regulate cardiovascular homeostasis, systemic metabolism, and inflammation. Therefore, through its endocrine function, the heart can maintain physiological conditions and prevent organ damage under pathological conditions.

Isotactic poly(4-methyl-1-pentene) melt as a porous liquid: Reduction of compressibility due to penetration of pressure medium.

A pressure-induced structural change of a polymer isotactic poly(4-methyl-1-pentene) (P4MP1) in the melted state at 270 °C has been investigated by high-pressure in situ x-ray diffraction, where high pressures up to 1.8 kbar were applied using helium gas. The first sharp diffraction peak (FSDP) position of the melt shows a less pressure dependence than that of the normal compression using a solid pressure transmitting medium. The contraction using helium gas was about 10% at 2 kbar, smaller than about 20% at the same pressure using a solid medium. The result indicates that helium entered the interstitial space between the main chains. The helium/monomer molar ratio was estimated to be 0.3 at 2 kbar from the FSDP positions. These results suggest that the compressibility of the P4MP1 melt can be largely dependent on the pressure transmitting media. As the pore size is reversibly and continuously controllable by compression, we suggest that the P4MP1 melt can be an ideal porous liquid for investigating a novel mechanical response of the pores in a non-crystalline substance.

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Development of a Video-Based Tool for the Self-Assessment of Eating Rates in Young Adult Females.

Eating speed assessments are often based on self-reports, and establishing objective "ratings" is required to improve accuracy. However, no relevant simple assessment tool incorporating "ratings" is currently available. This study aimed to develop a group-adaptable eating rate assessment tool for young females using smartphones. Fifty female college students were directly observed while eating, and a self-assessment tool for the eating rate was created using video. Using the directly observed eating rate of a test food A as the gold standard (GS), we compared the eating rate self-assessment findings between those obtained using a conventional questionnaire and those obtained using an assessment tool. The validity and reproducibility of the assessment tool were verified. In terms of validity, the correlation coefficient for the GS questionnaire (r=0.442, p<0.001) was similar to that for the self-assessment tool (r=0.491, p<0.001). The reproducibility of repeated measurements of the self-assessment tool was inferior to that of the questionnaire (weighted kappa coefficients; 0.393 vs. 0.804). This may be explained in part by participants selecting the same items with specific words such as "fast" or "slow" on two occasions. As for the validity of test food A, additional measurements for test food C on a subset of subjects (n=16) showed a strong positive correlation (r=0.845, p<0.001) between A and C. The present study suggests that a video-based self-assessment tool we developed for young adult females is straightforward, and allows the subjects to observe specific and visual ratings in a manner that is less burdensome and time-effective than conventional questionnaire methods.

Zebrafish Vascular Development: General and Tissue-Specific Regulation.

Circulation is required for the delivery of oxygen and nutrition to tissues and organs, as well as waste collection. Therefore, the heart and vessels develop first during embryogenesis. The circulatory system consists of the heart, blood vessels, and blood cells, which originate from the mesoderm. The gene expression pattern required for blood vessel development is predetermined by the hierarchical and sequential regulation of genes for the differentiation of mesodermal cells. Herein, we review how blood vessels form distinctly in different tissues or organs of zebrafish and how vessel formation is universally or tissue-specifically regulated by signal transduction pathways and blood flow. In addition, the unsolved issues of mutual contacts and interplay of circulatory organs during embryogenesis are discussed.

Mechanical load regulates bone growth via periosteal Osteocrin.

Mechanical stimuli including loading after birth promote bone growth. However, little is known about how mechanical force triggers biochemical signals to regulate bone growth. Here, we identified a periosteal-osteoblast-derived secretory peptide, Osteocrin (OSTN), as a mechanotransducer involved in load-induced long bone growth. OSTN produced by periosteal osteoblasts regulates growth plate growth by enhancing C-type natriuretic peptide (CNP)-dependent proliferation and maturation of chondrocytes, leading to elongation of long bones. Additionally, OSTN cooperates with CNP to regulate bone formation. CNP stimulates osteogenic differentiation of periosteal osteoprogenitors to induce bone formation. OSTN binds to natriuretic peptide receptor 3 (NPR3) in periosteal osteoprogenitors, thereby preventing NPR3-mediated clearance of CNP and consequently facilitating CNP-signal-mediated bone growth. Importantly, physiological loading induces Ostn expression in periosteal osteoblasts by suppressing Forkhead box protein O1 (FoxO1) transcription factor. Thus, this study reveals a crucial role of OSTN as a mechanotransducer converting mechanical loading to CNP-dependent bone formation.

Mapping of rice Ur1 (Undulated rachis-1) gene with effect on increasing spikelet number per panicle and sink size, and development of selection markers for the breeding by the use of Ur1.

Ur1 enhances secondary rachis-branching, resulting in more spikelets per panicle. This genic effect can increase grain yield by enlarging sink size. We conducted mapping of Ur1 using SSR markers, and detected markers usable for the MAS (marker-assisted selection) for Ur1. Three Ur1 isogenic lines of recurrent parents Taichung 65, 'Shiokari' and 'Nishihikari'('T(U)', 'S(U)' and 'N(U)', respectively) were used. SSR-marker analysis indicated that each isogenic line had a non-substituted region containing Ur1 on chromosome 6 which was inherited from its donor parent. T(U) was crossed with a non-Ur1-carrying line, and the F(2) and F(3) populations were grown. Recombination values between the Ur1 locus and SSR-marker loci were obtained from data of the F(2) and F(3). On the basis of both the linkage relationship and the non-substituted regions in T(U), S(U) and N(U), candidate region of the Ur1 locus was narrowed to 0.139 Mb between the loci of up85938 and SSR17 on the long arm of chromosome 6. Genotypes of T(U) and other five Ur1-carrying lines at each locus of ten SSR markers near the Ur1 locus were determined, and allelic frequency at each locus was investigated for 27 japonica and 21 indica varieties. Consequently, SSR12 and SSR17 could be employed as MAS markers for Ur1.

Pressure dependence of the structure of liquid copper halides.

X-ray diffraction of liquid CuI, CuBr and CuCl has been measured up to 19 GPa using synchrotron radiations. Static structure factor S(Q) and pair distribution function g(r) were obtained. For liquid CuI, CuBr and CuCl, S(Q) and g(r) change their shapes continuously with increasing pressure, indicating anisotropic compression of the local structures. The pressure dependence of the peak position ratio r(2)/r(1) of g(r) shows that Cu atoms are located in a tetrahedral site at low pressures and then in an octahedral site at high pressures in these liquid copper halides. At higher pressures, the local structure of liquid CuCl is similar to the intermediate structure between NaCl and CsCl structures. These results are compared with the pressure-induced structural transformation in the crystalline phase.

Intensity distribution profile of double Bragg scattering in the small-angle region from highly oriented pyrolytic graphite.

It is observed that radial streak patterns of double Bragg scattering appear in the small-angle X-ray scattering from highly oriented pyrolytic graphite (HOPG). The intensity profile of double Bragg scattering from an HOPG sample is calculated theoretically. Assuming that the c axes of the graphite crystallites in the HOPG sample are distributed around an orientation vector and their distribution function has a Gaussian form, it is found that the intensity profile of double Bragg scattering is expressed by a double Gaussian function of the scattering angle and the azimuthal angle of the streak. The calculated intensity profile is compared with the experimental one. The method developed in this article can be used to estimate the orientational distribution of crystallites in uniaxial polycrystalline materials.

Spatial Allocation and Specification of Cardiomyocytes during Zebrafish Embryogenesis.

Incomplete development and severe malformation of the heart result in miscarriage of embryos because of its malfunction as a pump for circulation. During cardiogenesis, development of the heart is precisely coordinated by the genetically-primed program that is revealed by the sequential expression of transcription factors. It is important to investigate how spatial allocation of the heart containing cardiomyocytes and other mesoderm-derived cells is determined. In addition, the molecular mechanism underlying cardiomyocyte differentiation still remains elusive. The location of ectoderm-, mesoderm-, and endoderm-derived organs is determined by their initial allocation and subsequent mutual cell-cell interactions or paracrine-based regulation. In the present work, we provide an overview of cardiac development controlled by the germ layers and discuss the points that should be uncovered in future for understanding cardiogenesis.

In Vivo Visualization of Cardiomyocyte Apicobasal Polarity Reveals Epithelial to Mesenchymal-like Transition during Cardiac Trabeculation.

Despite great strides in understanding cardiac trabeculation, many mechanistic aspects remain unclear. To elucidate how cardiomyocyte shape changes are regulated during this process, we engineered transgenes to label their apical and basolateral membranes. Using these tools, we observed that compact-layer cardiomyocytes are clearly polarized while delaminating cardiomyocytes have lost their polarity. The apical transgene also enabled the imaging of cardiomyocyte apical constriction in real time. Furthermore, we found that Neuregulin signaling and blood flow/cardiac contractility are required for cardiomyocyte apical constriction and depolarization. Notably, we observed the activation of Notch signaling in cardiomyocytes adjacent to those undergoing apical constriction, and we showed that this activation is positively regulated by Neuregulin signaling. Inhibition of Notch signaling did not increase the percentage of cardiomyocytes undergoing apical constriction or of trabecular cardiomyocytes. These studies provide information about cardiomyocyte polarization and enhance our understanding of the complex mechanisms underlying ventricular morphogenesis and maturation.

S1P-Yap1 signaling regulates endoderm formation required for cardiac precursor cell migration in zebrafish.

To form the primary heart tube in zebrafish, bilateral cardiac precursor cells (CPCs) migrate toward the midline beneath the endoderm. Mutants lacking endoderm and fish with defective sphingosine 1-phosphate (S1P) signaling exhibit cardia bifida. Endoderm defects lead to the lack of foothold for the CPCs, whereas the cause of cardia bifida in S1P signaling mutants remains unclear. Here we show that S1P signaling regulates CPC migration through Yes-associated protein 1 (Yap1)-dependent endoderm survival. Cardia bifida seen in spns2 (S1P transporter) morphants and s1pr2 (S1P receptor-2) morphants could be rescued by endodermal expression of nuclear localized form of yap1. yap1 morphants had decreased expression of the Yap1/Tead target connective tissue growth factor a (Ctgfa) and consequently increased endodermal cell apoptosis. Consistently, ctgfa morphants showed defects of the endodermal sheet and cardia bifida. Collectively, we show that S1pr2/Yap1-regulated ctgfa expression is essential for the proper endoderm formation required for CPC migration.

Suppression of adenylyl cyclase-mediated cAMP production by plasma membrane associated cytoskeletal protein 4.1G.

It has been shown lately that activity of G protein-coupled receptors (GPCRs) is regulated by an array of proteins binding to carboxy (C)-terminus of GPCRs. Proteins of 4.1 family are subsets of subcortical cytoskeletal proteins and are known to stabilize cellular structures and proteins at the plasma membrane. One of the 4.1 family proteins, 4.1G has been shown to interact with the C-terminus of GPCRs and regulate intracellular distribution of the receptors, including parathyroid hormone (PTH)/PTH-related protein receptor (PTHR). PTHR is coupled to trimeric G proteins G(s) and G(q), which activate the adenylyl cyclase/cyclic AMP (cAMP) pathway and phospholipase C pathway, respectively. During the course of investigation of the role of 4.1G on adenylyl cyclase/cAMP signaling pathway, we found that 4.1G suppressed forskolin-induced cAMP production in cells. The cAMP accumulation induced by forskolin was decreased in HEK293 cells overexpressing 4.1G or increased in 4.1G-knockdown cells. Furthermore, PTH -(1-34)-stimulated cAMP production was also suppressed in the presence of exogenously expressed 4.1G despite its activity to increase the distribution of PTHR to the cell surface. In cells overexpressing FERM domain-deleted 4.1G, a mutant form of the protein deficient in plasma membrane distribution, neither forskolin-induced nor PTH -(1-34)-stimulated cAMP production was not altered. The suppression of the forskolin-induced cAMP production was observed even in membrane preparations of 4.1G-overexpressing cells. In 4.1G-knockdown HEK293 cells, plasma membrane distribution of adenylyl cyclase 6, one of the major subtypes of the enzyme in the cells, showed a slight decrease, in spite of the increased production of cAMP in those cells when stimulated by forskolin. Also, cytochalasin D treatment did not cause any influence on forskolin-induced cAMP production in HEK293 cells. These data indicate that plasma membrane-associated 4.1G regulates GPCR-mediated G(s) signaling by suppressing adenylyl cyclase-mediated cAMP production.